Periodic Reporting for period 1 - ChondroCONNECT (Development of a connexon-proteoliposome delivery system to ameliorate inflammatory and mechanical stress responses in the regeneration of osteoarthritic cartilage)
Berichtszeitraum: 2020-09-01 bis 2022-08-31
Degeneration of cartilage at joint surfaces due to injury, age or disease (e.g. osteoarthritis; OA) causes significant pain, disability and decreased quality of life. Current treatments fail to effectively repair cartilage and typically only manage symptoms and delay the degeneration that leads to OA and the need for joint replacement. OA affects over 500 million people worldwide and costs countries in the EU approximately 1-2 % of their Gross Domestic Product. Adult cartilage has a poor capacity to heal, which is limited further by the inflammatory and senescent cell environment present in the degenerative joint. Recent attention has focused on therapies that promote cartilage regeneration and simultaneously target key inflammatory mediators to inhibit their effect. This approach has the potential to break the vicious cycle of cartilage degeneration and allow the tissue to repair sufficiently to restore mechanical functionality.
This fellowship focused on one such emerging potential molecular target, connexin-43 (Cx43), a protein that forms channels capable of transporting small molecules across cell membranes. In OA cartilage, Cx43 protein expression is increased approximately 40-fold. The mechanisms of Cx43 upregulation in OA and its role in OA pathology are yet to be fully understood but both channel and non-channel Cx43 activities appear to be important regulators of cell behaviour. Increased Cx43 expression has been linked to increased spreading of pro-inflammatory signalling in OA, while non-channel Cx43 activity may be involved in transcriptional regulation of cartilage-relevant genes like N-cadherin and Twist1. The above findings point towards an important role for a Cx43-sensitive signalling axis in cartilage cell (chondrocyte) responses to proinflammatory cytokines and adoption of OA-like cell phenotypes.
Therefore, during this project, I aimed to investigate the role of Cx43 in chondrocyte responses to proinflammatory stimuli associated with OA at various levels, including gene expression, metabolic regulation, and channel and non-channel activity. I then aimed to identify key processes in these responses, and develop and test an siRNA-based therapeutic for the regeneration of cartilage tissue in a three-dimensional culture environment.
This fellowship focused on one such emerging potential molecular target, connexin-43 (Cx43), a protein that forms channels capable of transporting small molecules across cell membranes. In OA cartilage, Cx43 protein expression is increased approximately 40-fold. The mechanisms of Cx43 upregulation in OA and its role in OA pathology are yet to be fully understood but both channel and non-channel Cx43 activities appear to be important regulators of cell behaviour. Increased Cx43 expression has been linked to increased spreading of pro-inflammatory signalling in OA, while non-channel Cx43 activity may be involved in transcriptional regulation of cartilage-relevant genes like N-cadherin and Twist1. The above findings point towards an important role for a Cx43-sensitive signalling axis in cartilage cell (chondrocyte) responses to proinflammatory cytokines and adoption of OA-like cell phenotypes.
Therefore, during this project, I aimed to investigate the role of Cx43 in chondrocyte responses to proinflammatory stimuli associated with OA at various levels, including gene expression, metabolic regulation, and channel and non-channel activity. I then aimed to identify key processes in these responses, and develop and test an siRNA-based therapeutic for the regeneration of cartilage tissue in a three-dimensional culture environment.
At the outset of this fellowship, healthy and OA chondrocytes were characterised in culture. This analysis established that baseline expression of healthy anabolic markers was decreased and expression of OA-associated degenerative enzymes (catabolic enzymes) and inflammatory markers was increased in OA chondrocytes compared to healthy chondrocytes. Furthermore, when challenged with a proinflammatory cytokine (IL-1β), the response of OA cells was exaggerated and prolonged compared to healthy cells. Cx43 expression was increased in OA-cells and in response to IL-1β. Next, we determined that through small molecule inhibition of Cx43 channel activity, negative cell responses to IL-1β could be attenuated. This indicated a role for Cx43 in cell responses to cytokine stimulation, to investigate this further we then examined the effects of siRNA knockdown of Cx43. In culture, Cx43 knockdown was observed to increase the baseline expression of healthy chondrocyte markers and decrease the expression of catabolic enzymes. Furthermore, knockdown was able to attenuate responses to IL-1β. Interestingly, cell metabolic responses to IL-1β stimulation were observed to be divergent in cells treated with Cx43 channel inhibitor and Cx43 siRNA, indicating that alternative modes of action may be present.
In other systems Cx43 interacts with yes-associated protein (YAP), an important mechanosensitive molecule, sequestering it to the cytoplasm. Meanwhile, YAP has been demonstrated to be an important regulator of nuclear factor-κB (NF-κB) activity in chondrocytes, an important transcription factor in cell inflammatory responses. We determined that Cx43 interacts with YAP in chondrocytes at the protein level and that increased Cx43 expression coincided with decrease nuclear YAP and increased nuclear P65 (a subunit of NF-κB; nuclear translocation indicates activation).
With a view to developing these findings towards translation, we then optimised a non-viral cell penetrating peptide, the glycosaminoglycan enhanced-binding transfection (GET) peptide, for siRNA delivery. As mesenchymal stem cells (MSCs) are commonly used as a therapeutic cell population for cartilage regeneration, we confirmed that GET-siRNA nanoparticles were able to knockdown Cx43 expression in 3D chondrogenic pellet cultures of MSCs and chondrocytes. In either cell type, this knockdown did not negatively affect the expression of healthy chondrocyte markers and as seen in 2 D cultures, was able to attenuate responses to IL-1β, including increasing the presence of cartilage-like extracellular matrix production in these cultures.
Next, we aimed to combine this GET-siRNA technology with a regenerative collagen and hyaluronic scaffold to create an siRNA-activated scaffold system for cartilage regeneration. GET-siRNA nanoparticles were incorporated into regenerative scaffolds and were demonstrated to effectively knockdown Cx43 expression in both chondrocytes and MSCs. Like in previous pellet culture experiments, this knockdown was again effective at improving cartilage-like extracellular matrix deposition in the presence of IL-1β. The applicability of this system to then deliver this GET-siRNA cargo in vivo and to penetrate the dense extracellular matrix of cartilage tissue was tested through explant cartilage cultures. In these models, it was demonstrated that fluorescently-tagged GET-siRNA nanoparticles were able to penetrate into the cartilage tissue and co-localised with tissue resident chondrocytes.
In other systems Cx43 interacts with yes-associated protein (YAP), an important mechanosensitive molecule, sequestering it to the cytoplasm. Meanwhile, YAP has been demonstrated to be an important regulator of nuclear factor-κB (NF-κB) activity in chondrocytes, an important transcription factor in cell inflammatory responses. We determined that Cx43 interacts with YAP in chondrocytes at the protein level and that increased Cx43 expression coincided with decrease nuclear YAP and increased nuclear P65 (a subunit of NF-κB; nuclear translocation indicates activation).
With a view to developing these findings towards translation, we then optimised a non-viral cell penetrating peptide, the glycosaminoglycan enhanced-binding transfection (GET) peptide, for siRNA delivery. As mesenchymal stem cells (MSCs) are commonly used as a therapeutic cell population for cartilage regeneration, we confirmed that GET-siRNA nanoparticles were able to knockdown Cx43 expression in 3D chondrogenic pellet cultures of MSCs and chondrocytes. In either cell type, this knockdown did not negatively affect the expression of healthy chondrocyte markers and as seen in 2 D cultures, was able to attenuate responses to IL-1β, including increasing the presence of cartilage-like extracellular matrix production in these cultures.
Next, we aimed to combine this GET-siRNA technology with a regenerative collagen and hyaluronic scaffold to create an siRNA-activated scaffold system for cartilage regeneration. GET-siRNA nanoparticles were incorporated into regenerative scaffolds and were demonstrated to effectively knockdown Cx43 expression in both chondrocytes and MSCs. Like in previous pellet culture experiments, this knockdown was again effective at improving cartilage-like extracellular matrix deposition in the presence of IL-1β. The applicability of this system to then deliver this GET-siRNA cargo in vivo and to penetrate the dense extracellular matrix of cartilage tissue was tested through explant cartilage cultures. In these models, it was demonstrated that fluorescently-tagged GET-siRNA nanoparticles were able to penetrate into the cartilage tissue and co-localised with tissue resident chondrocytes.
This project targeted attenuation of proinflammatory signalling in OA as a strategy to improve cartilage regeneration and halt degenerative processes. For this, the potential OA-therapeutic target Connexin-43 (Cx43) was investigated. This work resulted in an increased understanding of the role Cx43 in chondrocyte responses to OA-associated stimuli and in maintaining healthy chondrocyte phenotypes. Furthermore, it was determined that Cx43 knockdown did not affect chondrogenic differentiation of MSCs in chondrogenic pellet or scaffold cultures and improved the phenotypes of both chondrocytes and MSCs in vitro. Therefore, Cx43 showed significant promise as a target for the attenuation of negative pathological responses to the proinflammatory environment of degenerative joints. Next, this project developed a cartilage regenerative scaffold functionalised to non-virally deliver siRNA through a cell penetrating peptide. The functionality of this siRNA-activated scaffold was demonstrated in vitro to improve cell phenotypes and healthy cartilage-like extracellular matrix production in both chondrocytes and mesenchymal stem cells even in the presence of pro-inflammatory cytokines. In conclusion, we demonstrated that Cx43 has significant effects on cell responses to proinflammatory stimuli relevant to OA and that through development of a regenerative scaffold-mediated siRNA delivery system we could effectively knockdown Cx43 expression and improve cell phenotypes and ECM production in vitro. This chondro-regenerative, siRNA-activated scaffold represents a new approach to halting and even reversing OA-associated cartilage degeneration.